Humans and other life forms are comprised of living cells. Among the mechanisms through which the cells of an organism communicate with each other and obtain information and stimuli from their environment is through cell membrane receptor molecules expressed on the cell surface. Many such receptors have been identified, characterized, and sometimes classified into major receptor superfamilies based on structural motifs and signal transduction features. Such families include (but are not limited to) ligand-gated ion channel receptors, voltage-dependent ion channel receptors, receptor tyrosine kinases, receptor protein tyrosine phosphatases, and G protein-coupled receptors. The receptors are a first essential link for translating an extracellular signal into a cellular physiological response.
G protein-coupled receptors (ie., GPCRs) form a vast superfamily of cell surface receptors which are characterized by an amino-terminal extracellular domain, a carboxy-terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times. Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors. These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains. The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream effector molecules.
The GPCRs bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons. Not surprisingly, GPCRs are important in the normal (and sometimes the aberrant) function of many cell types. See generally Strosberg, Eur. J. Biochem., 1991, 196, 1-10 and Bohm et al., Biochem J., 1997, 322, 1-18. When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine nucleotide-binding regulatory protein (G protein) that is coupled to the intracellular portion or region of the receptor. The G protein, in turn, transmits a signal to an effector molecule within the cell by either stimulating or inhibiting the activity of that effector molecule. These effector molecules include adenylate cyclase, phospholipases and ion channels. Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol. It is through this sequence of events that an extracellular ligand stimulus exerts intracellular changes through a G protein-coupled receptor. Each such receptor has its own characteristic primary structure, expression pattern, ligand binding profile, and intracellular effector system.
Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, for example by activating or antagonizing such receptors. For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically to enhance or inhibit the action of the ligand. Some G protein-coupled receptors have roles in disease pathogenesis (e.g., certain chemokine receptors that act as HIV co-receptors may have a role in AIDS pathogenesis), and are attractive targets for therapeutic intervention even in the absence of knowledge of the natural ligand of the receptor. Other receptors are attractive targets for therapeutic intervention by virtue of their expression pattern in tissues or cell types that are themselves attractive targets for therapeutic intervention. Examples of this latter category of receptors include receptors expressed in immune cells, which can be targeted to either inhibit autoimmune responses or to enhance immune responses to fight pathogens or cancer; and receptors expressed in the brain or other neural organs and tissues, which are likely targets in the treatment of schizophrenia, depression, bipolar disease, or other neurological disorders. This latter category of receptor is also useful as a marker for identifying and/or purifying (e.g., via fluorescence-activated cell sorting) cellular subtypes that express the receptor. Unfortunately, only a limited number of G protein receptors from the central nervous system (CNS) are known. Thus, a need exists for G protein-coupled receptors that have been identified and show promise as targets for therapeutic intervention in a variety of animals, including humans.
Insects are recognized as major pests in agriculture and in human domestic environments. Insects also parasitize animals and humans, being denoted as ectoparasites in such cases, causing morbidity and mortality. Insects also serve as vectors for the transmission of viral and parasitic diseases to plants, animals and humans. Thus, there is a continuing and compelling need to discover new methods for controlling insect populations and for repelling and/or killing pathogenic or pestiferous species. One way to control insect populations by killing or paralyzing insects is through the use of chemical agents, denoted as insecticides, that are selectively toxic to insects and potentially other invertebrates. Currently, insecticides have enormous value for the control of insects that are damaging to agricultural products, including crops and livestock. Insecticides are also used in human domestic situations, for the control of lawn and garden pests as well as insects that are damaging or annoying to humans, including stinging or biting insects, flies and cockroaches. Insecticides also have enormous value for the treatment or prevention of disease states caused by ectoparasites in livestock animals and pets, including fleas, lice, ticks, mites and biting flies. However, current chemicals used as insecticide are not optimal. Some have demonstrable toxicity for mammals, while resistance to some of them has arisen in certain target species. Therefore, there exists a need for new selective insecticides that have novel mechanisms of action.
Examples of insect GPCRs that have neuropeptide ligands are known (Li, et al., EMBO Journal, 1991, 10, 3221-3229; Li, et al., J. Biol. Chem. 1992, 267, 9-12; Monnier, et al., J. Biol. Chem., 1992, 267, 1298-1302; Vanden Broeck, et al., Int. Rev. Cytology, 1996, 164, 189-268; Guerrero, Peptides, 1997, 18, 1-5; Hauser, et al., J. Biol. Chem., 1997, 272, 1002-1010; Birgul et al., EMBO J. 1999, 18, 5892-5900; Torfs et al., J. Neurochem. 2000, 74, 2182-2189; and Hauser et al. Biochem. Biophys. Res. Comm. 1998, 249, 822-828), though none has yet been publicly reported as having been exploited for insecticide discovery.
A large family of peptides generally 4-12 amino acids in length typically found in invertebrate animals (e.g. insects) is a class of neuropeptides known as FMRFamide related peptides (i.e., FaRPs). The prototypical FMRFamide peptides are so named because of the “FMRF” consensus amino acid sequence at their C-termini, consisting generally of (F,Y)(M,V,I,L)R(F,Y)NH2. As neuropeptides, these molecules are involved in vital biological processes requiring controlled neuromuscular activity. Although some neurotransmitters and neuromodulators (including neuropeptides) have been shown to function as ligands for receptors, to date there has been no identification of a FaRP neuropeptide as a ligand of a GPCR.
The allatostatins are an important group of insect neurohormones controlling diverse functions including the synthesis of juvenile hormones known to play a central role in metamorphosis and reproduction in various insect species. The very first Drosophila allatostatin, Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (i.e., drostatin-3), was isolated from Drosophila head extracts (Birgulet al., The EMBO J., 1999, 18, 5892-5900). Recently, a Drosophila allatostatin preprophormone gene has been cloned which encodes four Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH2 (drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH2 (drostatin-2), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (drostatin-3) and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH2 (drostatin-4) (Lenz et al., Biochem. Biophys. Res. Comm. 2000, 273, 1126-1131). The first Drosophila allatostatin receptor was cloned by Birgul et al. and shown to be functionally activated by drostatin-3 via Gi/Go pathways (Birgul et al., EMBO J. 1999, 18, 5892-5900). A second putative Drosophila allatostatin receptor (i.e., DARII). has been recently cloned (Lenz et al., Biochem. Biophys. Res. Comm. 2000, 273, 571-577). The DARII receptor cDNA (accession No. AF253526) codes for a protein that is strongly related to the first Drosophila allatostatin receptor. However, to date no functional activation of DARII by allatostatins has been reported.
The sulfakinins are a family of insect Tyr-sulfated neuropeptides. They show sequence and functional (myotropic effects, stimulation of digestive enzyme release) similarity to the vertebrate peptides gastrin and cholecystokinin. A gene encoding two sulfakinins (also called drosulfakinins), DSKI [Phe-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-amide] <SEQ ID NO: 160> and DSKII [Gly-Gly-Asp-Asp-Gln-Phe-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-amide] <SEQ ID NO: 161>, has been identified in Drosophila melanogaster (Nichols, (Mol. Cell Neuroscience, 1992, 3, 342-347; Nichols et al., J. Biol. Chem. 1988, 263, 12167-12170). The C-terminal heptapeptide sequence, Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-amide <SEQ ID NO: 162>, is identical in all sulfakinin identified so far from insects that are widely separated in evolutionary terms. The conservation of the heptapeptide sequence, including the presence of the sulfated Tyr residue, in widely divergent insect taxa presumably reflects functional significance of this myotropic “active core” (Nachman & Holman, in Insect Neuropeptides; chemistry, biology and action, Menn, Kelly & Massler, Eds., 1991, pp. 194-214, American Chemical Society, Washington, D.C.). To our knowledge, to date no receptors for insect sulfakinins have been identified.